Transcription of lncRNA prt, clustered prt RNA sites for Mmi1 binding, and RNA polymerase II CTD phospho-sites govern the repression of pho1 gene expression under phosphate-replete conditions in fission yeast

Abstract

Expression of fission yeast Pho1 acid phosphatase is repressed during growth in phosphate-rich medium. Repression is mediated by transcription of the prt locus upstream of pho1 to produce a long noncoding (lnc) prt RNA. Repression is also governed by RNA polymerase II CTD phosphorylation status, whereby inability to place a Ser7-PO 4 mark (as in S7A) derepresses Pho1 expression, and inability to place a Thr4-PO 4 mark (as in T4A) hyper-represses Pho1 in phosphate replete cells. Here we find that basal pho1 expression from the prt-pho1 locus is inversely correlated with the activity of the prt promoter, which resides in a 110-nucleotide DNA segment preceding the prt transcription start site. CTD mutations S7A and T4A had no effect on the activity of the prt promoter or the pho1 promoter, suggesting that S7A and T4A affect post-initiation events in prt lncRNA synthesis that make it less and more repressive of pho1, respectively. prt lncRNA contains clusters of DSR (determinant of selective removal) sequences recognized by the YTH-domain-containing protein Mmi1. Altering the nucleobase sequence of two DSR clusters in the prt lncRNA caused hyper-repression of pho1 in phosphate replete cells, concomitant with increased levels of the prt transcript. The isolated Mmi1 YTH domain binds to RNAs with single or tandem DSR elements, to the latter in a noncooperative fashion. We report the 1.75 Å crystal structure of the Mmi1 YTH domain and provide evidence that Mmi1 recognizes DSR RNA via a binding mode distinct from that of structurally homologous YTH proteins that recognize m 6 Amodified RNA.

A long noncoding (lnc)RNA governs expression of the phosphate transporter Pho84 in fission yeast and has cascading effects on the flanking prt lncRNA and pho1 genes

“…3C). The consensus HomolD element 5'-CAGTCAC(A/G) functions as a Pol2 promoter signal in fission yeast genes encoding ribosomal proteins (19)(20)(21) and in the prt and nc-tgp1 lncRNA genes that control phosphate homeostasis (6,8 3A) and expression of the prt2•pho1 reporter RNA (Fig. 3B).…”

“…The phosphate regulon in the fission yeast Schizosaccharomyces pombe comprises three genes that specify, respectively, a cell surface acid phosphatase Pho1, an inorganic phosphate transporter Pho84, and a glycerophosphate transporter Tgp1 (2). Expression of pho1 and tgp1 is actively repressed during growth in phosphate-rich medium by the transcription in cis of a long noncoding (lnc) RNA from the 5' flanking genes prt and nc-tgp1 (3)(4)(5)(6)(7)(8). The prt lncRNA initiates 1147 nucleotides (nt) upstream of the pho1 mRNA transcription start site (Fig.…”

“…Transcription of the prt and nc-tgp1 lncRNAs is driven by distinctive bi-partite promoters, consisting of a HomolD box and a TATA box located sequentially within a 100-nt segment preceding the start sites (6,8). Mutations in the HomolD and TATA boxes of the prt or nc-tgp1 genes result in derepression of the downstream pho1 or tgp1 promoters in phosphate-replete cells.…”

“…The ensuing synthesis of the pho1 and tgp1 mRNAs (as well as their induction during phosphate starvation) depends on the DNAbinding transcription factor Pho7 (2,9), which recognizes a 12-nt sequence motif (5'-TCG(G/C)(A/T)xxTTxAA) present in the pho1 and tgp1 promoters (10). A simple model for the repressive arm of fission yeast phosphate homeostasis is that transcription of the upstream lncRNA interferes with expression of the downstream genes encoding Pho1 or Tgp1 by displacing Pho7 from the pho1 and tgp1 promoters (6)(7)(8).…”

“…By contrast, prevention of the Thr4-PO 4 mark hyper-represses pho1 under phosphate-rich conditions (5). Because such CTD mutations do not affect the activity of the prt or pho1 promoters per se, it is proposed that CTD status affects Pol2 termination during prt lncRNA synthesis, and thus the propensity to displace Pho7 from the pho1 promoter (6). Absence of CTD Ser7-PO 4 or limitation of Ser5-PO 4 elicits a similar de-repression of transcription from the tgp1 promoter in phosphate-replete cells, without affecting the activity of the nc-tgp1 promoter (8).…”